P330: PROTEOMIC PROFILING UNCOVERS THERAPEUTIC VULNERABILITIES FOR TCF3 TRANSLOCATED B-CELL ACUTE LYMPHOBLASTIC LEUKEMIAS
Authors: Rebecca Hasselmann, Lena Blümel, Flavia Bernardi, Daniel Picard, Jacob Torrejon-Diaz, Vera Jepsen, Jasmin Bartl, Nan Qin, Beat Bornhauser, Sanil Bhatia, Blerim Marovca, Veronique Marsaud, Florent Dingli, Damarys Loew, Martin Stanulla, Jean-Pierre Bourquin, Arndt Borkhardt, Marc Remke, Olivier Ayrault, Ute Fischer
Journal: HemaSphere (2023)

Background: B-cell acute lymphoblastic leukemia (B-ALL) is the most frequent form of childhood ALL. The disease divides into more than 20 subtypes and is characterized by subtype-defining genetic alterations, including aneuploidies or chromosomal translocations. TCF3, encoding a member of the E protein (class I) family of helix-loop-helix transcription factors, is a master regulator of hematopoietic development and is frequently rearranged in B-ALL. Depending on the involved fusion partner gene, the impact on the patient´s outcome can differ markedly. The translocation t(1;19)(q23;p13), for instance, resulting in the TCF3-PBX1 fusion occurs in about 5% of pediatric B-ALL and is associated with a favorable outcome. The TCF3-HLF fusion, in contrast, that is the molecular equivalent of the translocation t(17;19)(q22;p13) represents a rare genetic entity (~0.5% of pediatric ALL) and is associated with a poor prognosis. The genetic and transcriptomic landscapes of TCF3-HLF and TCF3-PBX1 positive B-ALL have already been characterized in our previous work, leading to the identification of novel therapeutic targets. The proteomic landscape of these subtypes, however, is still unknown. Aims: Proteins are the functional entities of biological processes. mRNA and protein expression is not necessarily coherent. Therefore, we aim to use proteomic profiling to highlight underappreciated molecular biological features unique to TCF3-HLF or TCF3-PBX1 positive B-ALL and to identify alternative therapeutic approaches. Methods: Using state-of-the-art mass spectrometry-based techniques, we comprehensively analyzed the proteome of leukemia samples from patients with TCF3-HLF (n=6) and TCF3-PBX1 (n=5) positive B-ALL after transplantation in NSG mice. This approach was complemented using our high-throughput drug screening pipeline in an unbiased approach and apoptosis assays to identify and validate potential drugs. Results: We quantified >6000 proteins, which allow a clear distinction between TCF3-HLF and TCF3-PBX1 positive leukemias by unsupervised clustering methods. Gene set enrichment analysis (GSEA) identified several gene sets enriched in either of the two subgroups. RNA biology, transcription, mitochondrial translation and cellular respiration were among the most prominent enriched gene sets for TCF3-HLF positive leukemias, while immune response/cell cycle, actin cytoskeleton, cell morphogenesis and RTK signaling emerged for the TCF3-PBX1 positive subtype. High-throughput drug screening using an in-house established drug library consisting of >600 FDA-approved or clinical trial phase I-IV anti-cancer drugs identified 109 drugs showing a differential response between TCF3-HLF and TCF3-PBX1 positive leukemic cells. Notably, we identified novel potential drug targets that also support the GSEA performed on the proteomic data. Among others, these include MDM2 and DNA/RNA synthesis for TCF3-HLF positive leukemic cells and microtubule/tubulin and CDK for TCF3-PBX1 positive leukemic cells. We confirmed these findings using two independent apoptosis assays. Additionally, we identified promising targets for drug discovery. BLK was the most significantly dysregulated protein for the TCF3-PBX1 positive subtype. We tested the effect of the first selective BLK inhibitor BLK-IN-2 and confirmed high sensitivity in BLKhigh leukemic cells, while BLKlow leukemic cells only showed little response. Summary/Conclusion: We suggest that exploring the protein expression landscape of genetic subtypes of B-ALL is a promising approach for the discovery of putative biomarkers and promising novel therapeutic strategies. Keywords: Proteomics, Acute lymphoblastic leukemia, ALL